KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, which is synonymous with 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid and "deaminated neuraminic acid") is a novel sialic acid first isolated from the polysialoglycoproteins of rainbow trout (Salmo gairdneri) eggs. (Nadano, D. et al., (1986) J. Biol. Chem. 261: 11550-11557; Iwasaki, M. et al, (1987) Biochemistry 26:1452-1457; Inoue, S. et al., (1988) Biochem. Biophys. Res. Commun. 153:172-176).
Subsequently, KDN-containing glycoconjugates have been discovered throughout nature including in the sperm of rainbow trout (Song, S. et al., (1991) J. Biol. Chem. 266:21929-21935), the jelly coat of the eggs of the newt Pleurodeles waltlii (Strecker, G. et al., (1992) FEBS Lett. 298:39-48) and the capsule of the bacterium Klebsiella ozaenae (Knirel, Y. A. et al., (1989) Carbohydr. Res. 188:145-155.
KDN-containing glycoconjugates are refractory to regular sialidases. FIG. 1c shows the specificities of KDN-cleaving sialidase (KDN-sialidase) and regular sialidase.
Li, et al., made a poster presentation of the presence of a KDN-cleaving sialidase in the liver of the loach, Misgurnus fossilis at the Rinshoken International Conference, Tokyo, Japan, Sep. 27-29, 1993. Li, et al., also presented the same material at the 22nd annual meeting of the Society for Complex Carbohydrates in Puerto Rico, Nov. 17-20, 1993, Glycobiology, volume 3, p. 525, 1993. Subsequently, Li, et al., reported the partial purification of KDN-cleaving sialidase from loach liver (Li, Y.-T. et al.,). Arch. Biochem. Biophys. (1994) 310:243-246, the contents of which are incorporated herein by reference.
A KDN-cleaving enzyme called KDNase has also been recently induced in a microorganism, Sphmgobacterium multivorum (Kitajima, K., et al., J. Biol. Chem. (1994) 269:21415-21419). A detailed method for the isolation of KDN-cleaving sialidase from mollusks has never been described. The KDN-cleaving sialidase is an indispensable tool for the detection and characterization of KDN-containing glycoconjugates and for studying their biological function. No convenient source and method are currently available for facile preparation of a KDN-cleaving sialidase.
We discovered that the hepatopancreases of mollusks are rich in KDN-containing sialidase. The present invention describes a method for facile isolation of KDN-cleaving sialidase from a mollusk, using as an example, a marine bivalve lamellibranch mollusk of the family Ostreidae, an oyster.